The long term objective of the proposed research is to understand the role of the plasma membrane (PM) proteins and the cytoskeleton in mediating the formation of apical and basolateral domains of the plasma membrane in cells that exhibit a transcellular polarity. Examples of such cells are epithelial cells which are bounded on one side by a fluid phase and on the other by an extracellular matrix. In vitro adhesion of HeLa cells (a transformed epithelium easily grown in suspension) to an extracellular matrix protein, gelatin, will be used as a model to study the formation of the apical and basolateral PM. In this system the initial stages in the formation of these PM domains are cell attachment and spreading. HeLa cells attached to gelatin coated culture dishes are induced to spread rapidly and form an apical PM exposed to the culture medium and a basal PM adjacent to the culture dish. The apical and basal domains can be isolated and redistribution of proteins between the domains during spreading can be quantitated. We have identified five gelatin receptors on the HeLa cell surface. The kinetics of cell attachment and spreading indicate that spreading is a cooperative process leading to the hypothesis that the mechanism for spreading is the segregation of the cell surface extracellular matrix receptors into the basal PM with subsequent clustering of the receptors into oligomers inducing them to bind to the cytoskeleton. The hypothesis will be tested by concentrating on several specific aims: 1, determine which of the known gelatin receptors mediate attachment and/or spreading; 2, determine which receptors redistribute and become segregated among the apical, basal and internal PM domains during cell adhesion; 3, determine if gelatin receptor clustering is correlated with cell spreading; 4, determine if receptor binding to the cytoskeleton is regulated by receptor clustering and/or covalent modification; 5, determine which cytoskeletal proteins bind to the gelatin receptors and; 6, determine if the receptors are transmembrane proteins present in oligomeric complexes and if they have common polypeptide fragments. Results from the specific aims will undoubtedly increase our understanding of the molecular mechanism of cell adhesion to an extracellular matrix and the formation of transcellular polarity.